Method and composition for enhancing transport across biological membranes

ABSTRACT

Methods and compositions for transporting drugs and macromolecules across biological membranes are disclosed. In one embodiment, the invention includes a method for enhancing transport of a selected compound across a biological membrane, wherein a biological membrane is contacted with a conjugate containing a biologically active agent that is covalently attached to a transport polymer. In one embodiment, the polymer consists of from 6 to 25 subunits, at least 50% of which contain a guanidino or amidino sidechain moiety. The polymer is effective to impart to the attached agent a rate of trans-membrane transport across a biological membrane that is greater than the rate of trans-membrane transport of the agent in nonconjugated form.

This application claims priority under 35 U.S.C. §120 to U.S.provisional application Ser. No. 60/047,345 filed on May 21, 1997, whichis incorporated herein by reference.

GOVERMENT INTEREST

This invention was made with the support of NIH grant number CA 65237.Accordingly, the U.S. Government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions that areeffective to enhance transport of biologically active agents, such asorganic compounds, polypeptides, oligosaccharides, nucleic acids, andmetal ions, across biological membranes.

References

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All references cited within this application are incorporated herein byreference.

BACKGROUND OF THE INVENTION

The plasma membranes of cells present a barrier to passage of manyuseful therapeutic agents. In general, a drug must be freely soluble inboth the aqueous compartments of the body and the lipid layers throughwhich it must pass, in order to enter cells. Highly charged molecules inparticular experience difficulty in passing across membranes. Manytherapeutic macromolecules such as peptides and oligonucleotides arealso particularly intractable to transmembrane transport. Thus, whilebiotechnology has made available a greater number of potentiallyvaluable therapeutics, bioavailability considerations often hinder theirmedicinal utility. There is therefore a need for reliable means oftransporting drugs, and particularly macromolecules, into cells.

Heretofore, a number of transporter molecules have been proposed toescort molecules across biological membranes. Ryser et al. (1979)teaches the use of high molecular weight polymers of lysine forincreasing transport of various molecules across cellular membranes,with very high molecular weights being preferred. Although the authorscontemplated polymers of other positively charged residues such asornithine and arginine, operativity of such polymers was not shown.

Frankel et al. (1991) reported that conjugating selected molecules tothe tat protein of HIV can increase cellular uptake of those molecules.However, use of the tat protein has certain disadvantages, includingunfavorable aggregation and insolubility properties.

Barsoum et al. (1994) and Fawell et al. (1994) proposed using shorterfragments of the tat protein containing the tat basic region (residues49-57 having the sequence RKKRRQRRR. Barsoum et al. noted thatmoderately long polyarginine polymers (MW 5000-15000 daltons) failed toenable transport of β-galactosidase across cell membranes (e.g., Barsoumon page 3), contrary to the suggestion of Ryser et al. (supra).

Other studies have shown that a 16 amino acid peptide-cholesterolconjugate derived from the Antennapedia homeodomain is rapidlyinternalized by cultured neurons (Brugidou et al., 1995). However,slightly shorter versions of this peptide (15 residues) are noteffectively taken up by cells (Derossi et al., 1994).

The present invention is based in part on the applicants' discovery thatconjugation of certain polymers composed of contiguous, highly basicsubunits, particularly subunits containing guanidyl or amidinylmoieties, to small molecules or macromolecules is effective tosignificantly enhance transport of the attached molecule acrossbiological membranes. Moreover, transport occurs at a rate significantlygreater than the transport rate provided by a basic HIV tat peptideconsisting of residues 49-57.

SUMMARY OF THE INVENTION

The present invention includes, in one aspect, a method for enhancingtransport of a selected compound across a biological membrane. In themethod, a biological membrane is contacted with a conjugate containing abiologically active agent that is covalently attached to at least onetransport polymer. The conjugate is effective to promote transport ofthe agent across the biological membrane at a rate that is greater thanthe trans-membrane transport rate of the biological agent innon-conjugated form.

In one embodiment, the polymer consists of from 6 to 25 subunits, atleast 50% of which contain a guanidino or amidino sidechain moiety,wherein the polymer contains at least 6, and more preferably, at least 7guanidino or amidino sidechain moieties. In another embodiment, thepolymer consists of from 6 to 20, 7 to 20, or 7 to 15 subunits. Morepreferably, at least 70% of the subunits in the polymer containguanidino or amidino sidechain moiety, and more preferably still, 90%.Preferably, no guanidino or amidino sidechain moiety is separated fromanother such moiety by more than one non-guanidino or non-amidinosubunit. In a more specific embodiment, the polymer contains at least 6contiguous subunits each containing either a guanidino or amidino group,and preferably at least 6 or 7 contiguous guanidino sidechain moieties.

In another embodiment, the transport polymer contains from 6 to 25contiguous subunits, from 7 to 25, from 6 to 20, or preferably from 7 to20 contiguous subunits, each of which contains a guanidino or amidinosidechain moiety, and with the optional proviso that one of thecontiguous subunits can contain a non-arginine residue to which theagent is attached.

In one embodiment, each contiguous subunit contains a guanidino moiety,as exemplified by a polymer containing at least six contiguous arginineresidues.

Preferably, each transport polymer is linear. In a preferred embodiment,the agent is attached to a terminal end of the transport polymer.

In another specific embodiment, the conjugate contains a singletransport polymer.

The transport-enhancing polymers are exemplified, in a preferredembodiment, by peptides in which arginine residues constitute thesubunits. Such a polyarginine peptide may be composed of either all D-,all L- or mixed D- and L-arginines, and may include additional aminoacids. More preferably, at least one, and preferably all of the subunitsare D-arginine residues, to enhance biological stability of the polymerduring transit of the conjugate to its biological target.

The method may be used to enhance transport of selected therapeuticagents across any of a number of biological membranes including, but notlimited to, eukaryotic cell membranes, prokaryotic cell membranes, andcell walls. Exemplary prokaryotic cell membranes include bacterialmembranes. Exemplary eukaryotic cell membranes of interest include, butare not limited to membranes of dendritic cells, epithelial cells,endothelial cells, keratinocytes, muscle cells, fungal cells, bacterialcells, plant cells, and the like.

According to a preferred embodiment of the invention, the transportpolymer of the invention has an apparent affinity (Km) that is at least10-fold greater, and preferably at least 100-fold greater, than theaffinity measured for tat(49-75) peptide by the procedure of Example 6when measured at room temperature (23° C.) or 37° C.

Biologically active agents (which encompass therapeutic agents) include,but are not limited to metal ions, which are typically delivered asmetal chelates; small organic molecules, such as anticancer (e.g.,taxane) and antimicrobial molecules (e.g., against bacteria or fungisuch as yeast); and macromolecules such as nucleic acids, peptides,proteins, and analogs thereof. In one preferred embodiment, the agent isa nucleic acid or nucleic acid analog, such as a ribozyme whichoptionally contains one or more 2′-deoxy nucleotide subunits forenhanced stability. Alternatively, the agent is a peptide nucleic acid(PNA). In another preferred embodiment, the agent is a polypeptide, suchas a protein antigen, and the biological membrane is a cell membrane ofan antigen-presenting cell (APC). In another embodiment, the agent isselected to promote or elicit an immune response against a selectedtumor antigen. In another preferred embodiment, the agent is a taxane ortaxoid anticancer compound. In another embodiment, the agent is anon-polypeptide agent, preferably a non-polypeptide therapeutic agent.In a more general embodiment, the agent preferably has a molecularweight less than 10 kDa.

The agent may be linked to the polymer by a linking moiety, which mayimpart conformational flexibility within the conjugate and facilitateinteractions between the agent and its biological target. In oneembodiment, the linking moiety is a cleavable linker, e.g., containing alinker group that is cleavable by an enzyme or by solvent-mediatedcleavage, such as an ester, amide, or disulfide group. In anotherembodiment, the cleavable linker contains a photocleavable group.

In a more specific embodiment, the cleavable linker contains a firstcleavable group that is distal to the biologically active agent, and asecond cleavable group that is proximal to the agent, such that cleavageof the first cleavable group yields a linker-agent conjugate containinga nucleophilic moiety capable of reacting intramolecularly to cleave thesecond cleavable group, thereby releasing the agent from the linker andpolymer.

In another embodiment, the invention can be used to screen a pluralityof conjugates for a selected biological activity, wherein the conjugatesare formed from a plurality of candidate agents. The conjugates arecontacted with a cell that exhibits a detectable signal upon uptake ofthe conjugate into the cell, such that the magnitude of the signal isindicative of the efficacy of the conjugate with respect to the selectedbiological activity. This method is particularly useful for testing theactivities of agents that by themselves are unable, or poorly able, toenter cells to manifest biological activity. In one embodiment, thecandidate agents are selected from a combinatorial library.

The invention also includes a conjugate library which is useful forscreening in the above method.

In another aspect, the invention includes a pharmaceutical compositionfor delivering a biologically active agent across a biological membrane.The composition comprises a conjugate containing a biologically activeagent covalently attached to at least one transport polymer as describedabove, and a pharmaceutically acceptable excipient. The polymer iseffective to impart to the agent a rate of trans-membrane transport thatis greater than the trans-membrane transport rate of the agent innon-conjugated form. The composition may additionally be packaged withinstructions for using it.

In another aspect, the invention includes a therapeutic method fortreating a mammalian subject, particularly a human subject, with apharmaceutical composition as above.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are plots of cellular uptake of certainpolypeptide-fluorescein conjugates containing tat basic peptide (49-57,SEQ ID NO:1), poly-Lys (K9, SEQ ID NO:2), and poly-Arg (R4-R9 and r4-r9,SEQ ID NO:3-8 and 12-17, respectively), as a function of peptideconcentration; FIG. 1C is a histogram of uptake levels of the conjugatesmeasured for conjugates at a concentration of 12.5 μM (Examples 2-3);

FIGS. 2A-2F show computer-generated images of confocal micrographs(Example 4) showing emitted fluorescence (2A-2C) and transmitted light(2D-2F) from Jurkat cells after incubation at 37° C. for 10 minutes with6.25 μM of tat(49-57) conjugated to fluorescein (panels A and D), a7-mer of poly-L-arginine (R7) labeled with fluorescein (panels B and E),or a 7-mer of poly-D-arginine (r7) labeled with fluorescein (panels Cand F);

FIG. 3 shows cellular uptake of certain poly-Arg-fluorescein conjugates(r9, R9, R15, R20, and R25, SEQ ID NO: 17 and 8-11, respectively) as afunction of conjugate concentration (Example 5);

FIG. 4 shows a histogram of cellular uptake of fluorescein-conjugatedtat(49-57), and poly-Arg-fluorescein conjugates (R9, R8, and R7,respectively) in the absence (four bars on left) and presence (four barson right) of 0.5% sodium azide (Example 7);

FIGS. 5A-5C show plots of uptake levels of selected polymer conjugates(K9, R9, r4, r5, r6, r7, r8 and r9) by bacterial cells as a function ofconjugate concentration; FIG. 5A compares uptake levels observed for R9and r9 conjugates as a function of conjugate concentration, whenincubated with E. coli HB 101 cells; FIG. 5B shows uptake levelsobserved for K9 and r4 to r9 conjugates when incubated with E. coli HB101 cells; FIG. 5C compares uptake levels of conjugates of r9 and K9when incubated with Strep. Bovis cells;

FIGS. 6A-6E show exemplary conjugates of the invention which containcleavable linker moieties;

FIGS. 6F and 6G show chemical structures and conventional numbering ofconstituent backbone atoms for paclitaxel and “TAXOTERE”;

FIG. 6H shows a general chemical structure and ring atom numbering fortaxoid compounds; and

FIG. 7 shows inhibition of secretion of gamma-interferon (γ-IFN) bymurine T cells as a function of concentration of a sense-PNA-r7conjugate (SEQ ID NO: 6 conjulated to), antisense PNA-r7 conjugate (SEQID NO: 6 conjulated to), and non-conjugated antisense PNA (SEQ ID NO:19), where the PNA sequences are based on a sequence from the gene forgamma-interferon.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The term “biological membrane” as used herein refers to alipid-containing barrier which separates cells or groups of cells fromthe extracellular space. Biological membranes include, but are notlimited to, plasma membranes, cell walls, intracellular organellemembranes, such as the mitochondrial membrane, nuclear membranes, andthe like.

The term “transmembrane concentration” refers to the concentration of acompound present on the side of a membrane that is opposite or “trans”to the side of the membrane to which a particular composition has beenadded. For example, when a compound is added to the extracellular fluidof a cell, the amount of the compound measured subsequently inside thecell is the transmembrane concentration of the compound.

“Biologically active agent” or “biologically active substance” refers toa chemical substance, such as a small molecule, macromolecule, or metalion, that causes an observable change in the structure, function, orcomposition of a cell upon uptake by the cell. Observable changesinclude increased or decreased expression of one or more mRNAs,increased or decreased expression of one or more proteins,phosphorylation of a protein or other cell component, inhibition oractivation of an enzyme, inhibition or activation of binding betweenmembers of a binding pair, an increased or decreased rate of synthesisof a metabolite, increased or decreased cell proliferation, and thelike.

The term “macromolecule” as used herein refers to large molecules (MWgreater than 1000 daltons) exemplified by, but not limited to, peptides,proteins, oligonucleotides and polynucleotides of biological orsynthetic origin.

“Small organic molecule” refers to a carbon-containing agent having amolecular weight (MW) of less than or equal to 1000 daltons.

The terms “therapeutic agent”, “therapeutic composition”, and“therapeutic substance” refer, without limitation, to any compositionthat can be used to the benefit of a mammalian species. Such agents maytake the form of ions, small organic molecules, peptides, proteins orpolypeptides, oligonucleotides, and oligosaccarides, for example.

The terms “non-polypeptide agent” and “non-polypeptide therapeuticagent” refer to the portion of a transport polymer conjugate that doesnot include the transport-enhancing polymer, and that is a biologicallyactive agent other than a polypeptide. An example of a non-polypeptideagent is an anti-sense oligonucleotide, which can be conjugated to apoly-arginine peptide to form a conjugate for enhanced delivery acrossbiological membranes.

The term “polymer” refers to a linear chain of two or more identical ornon-identical subunits joined by covalent bonds. A peptide is an exampleof a polymer that can be composed of identical or non-identical aminoacid subunits that are joined by peptide linkages.

The term “peptide” as used herein refers to a compound made up of asingle chain of D- or L-amino acids or a mixture of D- and L-amino acidsjoined by peptide bonds. Generally, peptides contain at least two aminoacid residues and are less than about 50 amino acids in length.

The term “protein” as used herein refers to a compound that is composedof linearly arranged amino acids linked by peptide bonds, but incontrast to peptides, has a well-defined conformation. Proteins, asopposed to peptides, generally consist of chains of 50 or more aminoacids.

“Polypeptide” as used herein refers to a polymer of at least two aminoacid residues and which contains one or more peptide bonds.“Polypeptide” encompasses peptides and proteins, regardless of whetherthe polypeptide has a well-defined conformation.

The terms “guanidyl”, “guanidinyl”, and “guanidino” are usedinterchangeably to refer to a moiety having the formula —HN═C(NH₂)NH(unprotonated form). As an example, arginine contains a guanidyl(guanidino) moiety, and is also referred to as2-amino-5-guanidinovaleric acid or α-amino-δ-guanidinovaleric acid.“Guanidinium” refers to the positively charged conjugate acid form.

“Amidinyl” and “amidino” refer to a moiety having the formula—C(═NH)(NH₂). “Amidinium” refers to the positively charged conjugateacid form.

The term “poly-arginine” or “poly-Arg” refers to a polymeric sequencecomposed of contiguous arginine residues; poly-L-arginine refers to allL-arginines; poly-D-arginine refers to all D-arginines. Poly-L-arginineis also abbreviated by an upper case “R” followed by the number ofL-arginines in the peptide (e.g., R8 represents an 8-mer of contiguousL-arginine residues); poly-D-arginine is abbreviated by a lower case “r”followed by the number of D-arginines in the peptide (r8 represents an8-mer of contiguous D-arginine residues).

Amino acid residues are referred to herein by their full names or bystandard single-letter or three-letter notations: A, Ala, alanine; C,Cys, cysteine; D, Asp, aspartic acid; E, Glu, glutamic acid; F, Phe,phenylalanine; G, Gly, glycine; H, His, histidine; I, Ile, isoleucine;K, Lys, lysine; L, Leu, leucine; M, Met, methionine; N, Asn, asparagine;P, Pro, proline; Q, Gln, glutamine; R, Arg, arginine; S, Ser, serine; T,Thr, threonine; V, Val, valine; W, Trp, tryptophan; X, Hyp,hydroxyproline; Y, Tyr, tyrosine.

II. Structure of Polymer Moiety

In one embodiment, transport polymers in accordance with the presentinvention contain short-length polymers of from 6 to up to 25 subunits,as described above. The conjugate is effective to enhance the transportrate of the conjugate across the biological membrane relative to thetransport rate of the non-conjugated biological agent alone. Althoughexemplified polymer compositions are peptides, the polymers may containnon-peptide backbones and/or subunits as discussed further below.

In an important aspect of the invention, the conjugates of the inventionare particularly useful for transporting biologically active agentsacross cell or organelle membranes, when the agents are of the type thatrequire trans-membrane transport to exhibit their biological effects,and that do not exhibit their biological effects primarily by binding toa surface receptor, i.e., such that entry of the agent does not occur.Further, the conjugates are particularly useful for transportingbiologically active agents of the type that require trans-membranetransport to exhibit their biological effects, and that by themselves(without conjugation to a transport polymer or some other modification),are unable, or only poorly able, to enter cells to manifest biologicalactivity.

As a general matter, the transport polymer used in the conjugatepreferably includes a linear backbone of subunits. The backbone willusually comprise heteroatoms selected from carbon, nitrogen, oxygen,sulfur, and phosphorus, with the majority of backbone chain atomsusually consisting of carbon. Each subunit contains a sidechain moietythat includes a terminal guanidino or amidino group.

Although the spacing between adjacent sidechain moieties will usually beconsistent from subunit to subunit, the polymers used in the inventioncan also include variable spacing between sidechain moieties along thebackbone.

The sidechain moieties extend away from the backbone such that thecentral guanidino or amidino carbon atom (to which the NH₂ groups areattached) is linked to the backbone by a sidechain linker thatpreferably contains at least 2 linker chain atoms, more preferably from2 to 5 chain atoms, such that the central carbon atom is the third tosixth chain atom away from the backbone. The chain atoms are preferablyprovided as methylene carbon atoms, although one or more other atomssuch as oxygen, sulfur, or nitrogen can also be present. Preferably, thesidechain linker between the backbone and the central carbon atom of theguanidino or amidino group is 4 chain atoms long, as exemplified by anarginine side chain.

The transport polymer sequence of the invention can be flanked by one ormore non-guanidino/non-amidino subunits, or a linker such as anaminocaproic acid group, which do not significantly affect the rate ofmembrane transport of the corresponding polymer-containing conjugate,such as glycine, alanine, and cysteine, for example. Also, any freeamino terminal group can be capped with a blocking group, such as anacetyl or benzyl group, to prevent ubiquitination in vivo.

The agent to be transported can be linked to the transport polymeraccording to a number of embodiments. In one preferred embodiment, theagent is linked to a single transport polymer, either via linkage to aterminal end of the transport polymer or to an internal subunit withinthe polymer via a suitable linking group.

In a second embodiment, the agent is attached to more than one polymer,in the same manner as above. This embodiment is somewhat less preferred,since it can lead to crosslinking of adjacent cells.

In a third embodiment, the conjugate contains two agent moietiesattached to each terminal end of the polymer. For this embodiment, it ispreferred that the agent has a molecular weight of less than 10 kDa.

With regard to the first and third embodiments just mentioned, the agentis generally not attached to one any of the guanidino or amidinosidechains so that they are free to interact with the target membrane.

The conjugates of the invention can be prepared by straightforwardsynthetic schemes. Furthermore, the conjugate products are usuallysubstantially homogeneous in length and composition, so that theyprovide greater consistency and reproducibility in their effects thanheterogenous mixtures.

According to an important aspect of the present invention, it has beenfound by the applicants that attachment of a single transport polymer toany of a variety of types of biologically active agents is sufficient tosubstantially enhance the rate of uptake of an agent across biologicalmembranes, even without requiring the presence of a large hydrophobicmoiety in the conjugate. In fact, attaching a large hydrophobic moietymay significantly impede or prevent cross-membrane transport due toadhesion of the hydrophobic moiety to the lipid bilayer. Accordingly,the present invention includes conjugates that do not contain largehydrophobic moieties, such as lipid and fatty acid molecules. In anotherembodiment, the method is used to treat a non-central nervous system(non-CNS) condition in a subject that does not require delivery throughthe blood brain barrier.

A. Polymer Components

Amino acids. In one embodiment, the transport polymer is composed of Dor L amino acid residues. Use of naturally occurring L-amino acidresidues in the transport polymers has the advantage that break-downproducts should be relatively non-toxic to the cell or organism.Preferred amino acid subunits are arginine (α-amino-δ-guanidinovalericacid) and α-amino-ε-amidinohexanoic acid (isosteric amidino analog). Theguanidinium group in arginine has a pKa of about 12.5.

More generally, it is preferred that each polymer subunit contains ahighly basic sidechain moiety which (i) has a pKa of greater than 11,more preferably 12.5 or greater, and (ii) contains, in its protonatedstate, at least two geminal amino groups (NH₂) which share aresonance-stabilized positive charge, which gives the moiety a bidentatecharacter.

Other amino acids, such as α-amino-β-guanidino-propionic acid,α-amino-γ-guanidinobutyric acid, or α-amino-ε-guanidinocaproic acid canalso be used (containing 2, 3 or 5 linker atoms, respectively, betweenthe backbone chain and the central guanidinium carbon).

D-amino acids may also be used in the transport polymers. Compositionscontaining exclusively D-amino acids have the advantage of decreasedenzymatic degradation. However, they may also remain largely intactwithin the target cell. Such stability is generally not problematic ifthe agent is biologically active when the polymer is still attached. Foragents that are inactive in conjugate form, a linker that is cleavableat the site of action (e.g., by enzyme- or solvent-mediated cleavagewithin a cell) should be included within the conjugate to promoterelease of the agent in cells or organelles.

Other Subunits. Subunits other than amino acids may also be selected foruse in forming transport polymers. Such subunits may include, but arenot limited to hydroxy amino acids, N-methyl-amino acids aminoaldehydes, and the like, which result in polymers with reduced peptidebonds. Other subunit types can be used, depending on the nature of theselected backbone, as discussed in the next section.

B. Backbone Type

A variety of backbone types can be used to order and position thesidechain guanidino and/or amidino moieties, such as alkyl backbonemoieties joined by thioethers or sulfonyl groups, hydroxy acid esters(equivalent to replacing amide linkages with ester linkages), replacingthe alpha carbon with nitrogen to form an aza analog, alkyl backbonemoieties joined by carbamate groups, polyethyleneimines (PEIs), andamino aldehydes, which result in polymers composed of secondary amines.

A more detailed backbone list includes N-substituted amide (CONRreplaces CONH linkages), esters (CO₂), keto-methylene (COCH₂) reduced ormethyleneamino (CH₂NH), thioamide (CSNH), phosphinate (PO₂RCH₂),phosphonamidate and phosphonamidate ester (PO2RNH), retropeptide (NHCO),transalkene (CR═CH), fluoroalkene (CF═CH), dimethylene (CH₂CH₂),thioether (CH₂S), hydroxyethylene (CH(OH)CH₂), methyleneoxy (CH₂O),tetrazole (CN₄), retrothioamide (NHCS), retroreduced (NHCH₂),sulfonamido (SO₂NH), methylenesulfonamido (CHRSO₂NH), retrosulfonamide(NHSO₂), and peptoids (N-substituted glycines), and backbones withmalonate and/or gem-diaminoalkyl subunits, for example, as reviewed byFletcher et al. (1998) and detailed by references cited therein. Peptoidbackbones (N-substituted glycines) can also be used (e.g., Kessler,1993; Zuckermann et al., 1992; and Simon et al., 1992). Many of theforegoing substitutions result in approximately isosteric polymerbackbones relative to backbones formed from α-amino acids.

Studies carried out in support of the present invention have utilizedpolypeptides (e.g., peptide backbones). However, other backbones, suchas those described above, may provide enhanced biological stability (forexample, resistance to enzymatic degradation in vivo).

C. Synthesis of Polymeric Transport Molecules

Polymers are constructed by any method known in the art. Exemplarypeptide polymers can be produced synthetically, preferably using apeptide synthesizer (Applied Biosystems Model 433) or can be synthesizedrecombinantly by methods well known in the art. Recombinant synthesis isgenerally used when the transport polymer is a peptide which is fused toa polypeptide or protein of interest.

N-methyl and hydroxy-amino acids can be substituted for conventionalamino acids in solid phase peptide synthesis. However, production ofpolymers with reduced peptide bonds requires synthesis of the dimer ofamino acids containing the reduced peptide bond. Such dimers areincorporated into polymers using standard solid phase synthesisprocedures. Other synthesis procedures are well known and can be found,for example, in Fletcher et al. (1998), Simon et al. (1992), andreferences cited therein.

III. Attachment of Transport Polymers To Biologically Active Agents

Transport polymers of the invention can be attached covalently tobiologically active agents by chemical or recombinant methods.

A. Chemical Linkages

Biologically active agents such as small organic molecules andmacromoles can be linked to transport polymers of the invention via anumber of methods known in the art (see, for example, Wong, 1991),either directly (e.g., with a carbodiimide) or via a linking moiety. Inparticular, carbamate, ester, thioether, disulfide, and hydrazonelinkages are generally easy to form and suitable for most applications.Ester and disulfide linkages are preferred if the linkage is to bereadily degraded in the cytosol, after transport of the substance acrossthe cell membrane.

Various functional groups (hydroxyl, amino, halogen, etc.) can be usedto attach the biologically active agent to the transport polymer. Groupswhich are not known to be part of an active site of the biologicallyactive agent are preferred, particularly if the polypeptide or anyportion thereof is to remain attached to the substance after delivery.

Polymers, such as peptides produced according to Example 1, aregenerally produced with an amino terminal protecting group, such asFMOC. For biologically active agents which can survive the conditionsused to cleave the polypeptide from the synthesis resin and deprotectthe sidechains, the FMOC may be cleaved from the N-terminus of thecompleted resin-bound polypeptide so that the agent can be linked to thefree N-terminal amine. In such cases, the agent to be attached istypically activated by methods well known in the art to produce anactive ester or active carbonate moiety effective to form an amide orcarbamate linkage, respectively, with the polymer amino group. Ofcourse, other linking chemistries can also be used.

To help minimize side-reactions, guanidino and amidino moieities can beblocked using conventional protecting groups, such as carbobenzyloxygroups (CBZ), di-t-BOC, PMC, Pbf, N-NO2, and the like.

Coupling reactions are performed by known coupling methods in any of anarray of solvents, such as N,N-dimethyl formamide (DMF), N-methylpyrrolidinone, dichloromethane, water, and the like. Exemplary couplingreagents include O-benzotriazolyloxy tetramethyluroniumhexafluorophosphate (HATU), dicyclohexyl carbodiimide, bromo-tris(pyrrolidino) phosphonium bromide (PyBroP), etc. Other reagents can beincluded, such as N,N-dimethylamino pyridine (DMAP), 4-pyrrolidinopyridine, N-hydroxy succinimide, N-hydroxy benzotriazole, and the like.

For biologically active agents that are inactive until the attachedtransport polymer is released, the linker is preferably a readilycleavable linker, meaning that it is susceptible to enzymatic orsolvent-mediated cleavage in vivo. For this purpose, linkers containingcarboxylic acid esters and disulfide bonds are preferred, where theformer groups are hydrolyzed enzymatically or chemically, and the latterare severed by disulfide exchange, e.g., in the presence of glutathione.

In one preferred embodiment, the cleavable linker contains a firstcleavable group that is distal to the agent, and a second cleavablegroup that is proximal to the agent, such that cleavage of the firstcleavable group yields a linker-agent conjugate containing anucleophilic moiety capable of reacting intramolecularly to cleave thesecond cleavable group, thereby releasing the agent from the linker andpolymer. This embodiment is further illustrated by the various smallmolecule conjugates discussed below.

B. Fusion Polypeptides

Transport peptide polymers of the invention can be attached tobiologically active polypeptide agents by recombinant means byconstructing vectors for fusion proteins comprising the polypeptide ofinterest and the transport peptide, according to methods well known inthe art. Generally, the transport peptide component will be attached atthe C-terminus or N-terminus of the polypeptide of interest, optionallyvia a short peptide linker.

IV. Enhanced Transport of Biologically Active Agents Across BiologicalMembranes

A. Measuring Transport Across Biological Membranes

Model systems for assessing the ability of polymers of the invention totransport biomolecules and other therapeutic substances acrossbiological membranes include systems that measure the ability of thepolymer to transport a covalently attached fluorescent molecule acrossthe membrane. For example, fluorescein (∞376 MW) can serve as a modelfor transport of small organic molecules (Example 2). For transport ofmacromolecules, a transport polymer can be fused to a large polypeptidesuch as ovalbumin (molecular weight 45 kDa; e.g., Example 14). Detectinguptake of macromolecules may be facilitated by attaching a fluorescenttag. Cellular uptake can also be analyzed by confocal microscopy(Example 4).

B. Enhanced Transport Across Biological Membranes

In experiments carried out in support of the present invention,transmembrane transport and concomitant cellular uptake was assessed byuptake of a transport peptide linked to fluorescein, according tomethods described in Examples 2 and 3. Briefly, suspensions of cellswere incubated with fluorescent conjugates suspended in buffer forvarying times at 37° C., 23° C., or 3° C. After incubation, the reactionwas stopped and the cells were collected by centrifugation and analyzedfor fluorescence using fluorescence-activated cell sorting (FACS).

Under the conditions used, cellular uptake of the conjugates was notsaturable. Consequently, ED₅₀ values could not be calculated for thepeptides. Instead, data are presented as histograms to allow directcomparisons of cellular uptake at single conjugate concentrations.

FIGS. 1A-1C show results from a study in which polymers of L-arginine(R; FIG. 1A) or D-arginine (r; FIG. 1B) ranging in length from 4 to 9arginine subunits were tested for ability to transport fluorescein intoJurkat cells. For comparison, transport levels for an HIV tat residues49-57 (“49-57”) and a nonamer of L-lysine (K9) were also tested. FIG. 1Cshows a histogram of uptake levels for the conjugates at a concentrationof 12.5 μM.

As shown in the figures, fluorescently labeled peptide polymers composedof 6 or more arginine residues entered cells more efficiently than thetat sequence 49-57. In particular, uptake was enhanced to at least abouttwice the uptake level of tat 49-57, and as much as about 6-7 times theuptake level of tat 49-57. Uptake of fluorescein alone was negligible.Also, the lysine nonamer (K9) showed very little uptake, indicating thatshort lysine polymers are ineffective as trans-membrane transports, incontrast to comparable-length guanidinium-containing polymers.

With reference to FIG. 1B, homopolymers of D-arginine exhibited evengreater transport activity than the L-counterparts. However, the orderof uptake levels was about the same. For the D-homopolymers, thepeptides with 7 to 9 arginines exhibited roughly equal activity. Thehexamer (R6 or r6) was somewhat less effective, but still exhibited atleast about 2 to 3-fold higher transport activity than tat(49-57).

The ability of the D- and L-arginine polymers to enhance trans-membranetransport was confirmed by confocal icroscopy (FIGS. 2A-2F and Example4). Consistent with the FACS data described above, the cytosol of cellsincubated with either R9 (FIGS. 2B and 2E) or r9 (FIGS. 2C and 2F) wasbrightly fluorescent, indicating high levels of conjugate transport intothe cells. In contrast, tat(49-57) at the same concentration showed onlyweak staining (FIGS. 2A and 2D). The confocal micrographs also emphasizethe point that the D-arginine polymer (FIG. 2C) was more effective atentering cells than the polymer composed of L-arginine (FIG. 2F).

From the foregoing, it is apparent that transport polymers of theinvention are significantly more effective than HIV tat peptide 47-59 intransporting drugs across the plasma membranes of cells. Moreover, thepoly-Lys nonamer was ineffective as a transporter.

To determine whether there was an optimal length for contiguousguanidinium-containing homopolymers, a set of longer argininehomopolymer conjugates (R15, R20, R25, and R30) were examined. Toexamine the effect of substantially longer polymers, a mixture ofL-arginine polymers with an average molecular weight of 12,000 daltons(∞100 amino acids) was also tested (Example 5). However, to avoidprecipitation problems, the level of serum in the assay had to bereduced for testing conjugates with the ∞12,000 MW polymer material.Cell uptake was analyzed by FACS as above, and the mean fluorescence oflive cells was measured. Cytotoxicity of each conjugate was alsomeasured.

With reference to FIG. 3, uptake of L-arginine homopolymer conjugateswith 15 or more arginines exhibited patterns of cellular uptakedistinctly different from polymers containing nine arginines or less.The curves of the longer conjugates were flatter, crossing those of theR9 and r9 conjugates. At higher concentrations (>3 μM), uptake of R9 andr9 was significantly better than for the longer polymers. However, atlower concentrations, cells incubated with the longer peptides exhibitedgreater fluorescence.

Based on this data, it appears that r9 and R9 enter the cells at higherrates than polymers containing 15 or more contiguous arginines. However,the biological half-life of R9 (L-peptide) was shorter than for thelonger conjugates, presumably because proteolysis of the longer peptides(due to serum enzymes) produces fragments that retain transportactivity. In contrast, the D-isomer (r9) did not show evidence ofproteolytic degradation, consistent with the high specificity of serumproteases for L-polypeptides.

Thus, overall transport efficacy of a transport polymer appears todepend on a combination of (i) rate of trans-membrane uptake (polymerwith less than about 15 continuous arginines are better) versussusceptibility to proteolytic inactivation (longer polymers are better).Accordingly, polymers containing 7 to 20 contiguous guanidiniumresidues, and preferably 7 to 15, are preferred.

Notably, the high molecular weight polyarginine conjugate (12,000 MW)did not exhibit detectable uptake. This result is consistent with theobservations of Barsoum et al. (1994), and suggests that argininepolymers have transport properties that are significantly different fromthose that may be exhibited by lysine polymers. Furthermore, the 12,000polyarginine conjugate was found to be highly toxic (Example 5). Ingeneral, toxicity of the polymers increased with length, though only the12,000 MW conjugate showed high toxicity at all concentrations tested.

When cellular uptake of polymers of D- and L-arginine were analyzed byMichaelis-Menten kinetics (Example 6), the rate of uptake by Jurkatcells was so efficient that precise K_(m) values could only be obtainedwhen the assays were carried out at 3° C. (on ice). Both the maximalrate of transport (V_(max)) and the apparent affinity of the peptidesfor the putative receptor of the Michaelis constant (K_(m)) were derivedfrom Lineweaver-Burk plots of the observed fluorescence of Jurkat cellsafter incubation with varying concentrations of nonamers of D- andL-arginine for 30, 60, 120, and 240 seconds.

Kinetic analysis also reveals that polymers rich in arginine exhibit abetter ability to bind to and traverse a putative cellular transportsite than, for example, the tat(49-57) peptide, since the K_(m) fortransport of the nonameric poly-L-arginine (44 μM) was substantiallylower than the K_(m) of the tat peptide (722 μM). Moreover, the nonamerof D-arginine exhibited the lowest Km (7 μM) of the polymers tested inthis assay (Table 1), i.e., an approximately 100-fold greater apparentaffinity.

According to a preferred embodiment of the invention, the transportpolymer of the invention has an apparent affinity (Km) that is at least10-fold greater, and preferably at least 100-fold greater, than theaffinity measured for tat by the procedure of Example 6 when measured atroom temperature (23° C.) or 37° C.

TABLE 1 K_(M) (μM) V_(MAX) (μM/sec) H₃N-RRRRRRRRR-COO⁻ 44.43 0.35H₃N-rrrrrrrrr-COO⁻ 7.21 0.39 tat 49-57 722 0.38

Experiments carried out in support of the present invention indicatethat polymer-facilitated transport is dependent upon metabolic integrityof cells. Addition of a toxic amount of sodium azide (0.5% w/v) to cellsresulted in inhibition of uptake of conjugates by about 9% (Example 7).The results shown in FIG. 4 demonstrate (i) sodium azide sensitivity oftrans-membrane transport, suggesting energy-dependence (cellularuptake), and (ii) the superiority of poly-guanidinium polymers of theinvention (R9, R8, R7) relative to HIV tat(49-57).

Without ascribing to any particular theory, the data suggest that thetransport process is an energy-dependent process mediated by specificrecognition of guanidinium or amidinium-containing polymers by amolecular transporter present in cellular plasma membranes.

Other experiments in support of the invention have shown that theconjugates of the invention are effective to transport biologicallyactive agents across membranes of a variety of cell types, includinghuman T cells (Jurkat), B cells (murine CH27), lymphoma T cells (murineEL-4), mastocytoma cells (murine P388), several murine T cellhybridomas, neuronal cells (PC-12), fibroblasts (murine RT), kidneycells (murine HELA), myeloblastoma (murine K562); and primary tissuecells, including all human blood cells (except red blood cells), such asT and B lymphocytes, macrophages, dendritic cells, and eosinophils;basophiles, mast cells, endothelial cells, cardiac tissue cells, livercells, spleen cells, lymph node cells, and keratinocytes.

The conjugates are also effective to traverse both gram negative andgram positive bacterial cells, as disclosed in Example 8 and FIGS.5A-5C. In general, polymers of D-arginine subunits were found to enterboth gram-positive and gram-negative bacteria at rates significantlyfaster than the transport rates observed for polymers of L-arginine.This is illustrated by FIG. 5A, which shows much higher uptake levelsfor r9 conjugate (D-arginines), than for the R9 conjugate (L-arginines),when incubated with E. coli HB 101 (prokariotic) cells. This observationmay be attributable to proteolytic degradation of the L-polymers bybacterial enzymes.

FIG. 5B shows uptake levels for D-arginine conjugates as a function oflength (r4 to r9) in comparison to a poly-L-lysine conjugate (K9), whenincubated with E. coli HB 101 cells. As can be seen, the polyarginineconjugates showed a trend similar to that in FIG. 2B observed witheukariotic cells, such that polymers shorter than r6 showed low uptakelevels, with uptake levels increasing as a function of length.

Gram-positive bacteria, as exemplified by Strep. bovis, were alsostained efficiently with polymers of arginine, but not lysine, as shownin FIG. 5C.

More generally, maximum uptake levels by the bacteria were observed at37° C. However, significant staining was observed when incubation wasperformed either at room temperature or at 3° C. Confocal microscopyrevealed that pretreatment of the bacteria with 0.5% sodium azideinhibited transport across the inner plasma membranes of bothgram-positive and gram-negative bacteria, but not transport across thecell wall (gram-positive bacteria) into the periplasmic space.

Thus, the invention includes conjugates that contain antimicrobialagents, such as antibacterial and antifungal compounds, for use inpreventing or inhibiting microbial proliferation or infection, and fordisinfecting surfaces to improve medical safety. In addition, theinvention can be used for transport into plant cells, particularly ingreen leafy plants.

Additional studies in support of the invention have shown thattranslocation across bacterial membranes is both energy- andtemperature-dependent, consistent with observations noted earlier forother cell-types.

V. Therapeutic Compositions

A. Small Organic Molecules

Small organic molecule therapeutic agents may be advantageously attachedto linear polymeric compositions as described herein, to facilitate orenhance transport across biological membranes. For example, delivery ofhighly charged agents, such as levodopa (L-3,4-dihydroxy-phenylalanine;L-DOPA) may benefit by linkage to polymeric transport molecules asdescribed herein. Peptoid and peptidomimetic agents are alsocontemplated (e.g., Langston, 1997; Giannis et al., 1997). Also, theinvention is advantageous for delivering small organic molecules thathave poor solubilities in aqueous liquids, such as serum and aqueoussaline. Thus, compounds whose therapeutic efficacies are limited bytheir low solubilities can be administered in greater dosages accordingto the present invention, and can be more efficacious on a molar basisin conjugate form, relative to the non-conjugate form, due to higheruptake levels by cells.

Since a significant portion of the topological surface of a smallmolecule is often involved, and therefore required, for biologicalactivity, the small molecule portion of the conjugate in particularcases may need to be severed from the attached transport polymer andlinker moiety (if any) for the small molecule agent to exert biologicalactivity after crossing the target biological membrane. For suchsituations, the conjugate preferably includes a cleavable linker forreleasing free drug after passing through a biological membrane.

In one approach, the conjugate can include a disulfide linkage, asillustrated in FIG. 6A, which shows a conjugate (I) containing atransport polymer T which is linked to a cytotoxic agent,6-mercaptopurine, by an N-acetyl-protected cysteine group which servesas a linker. Thus, the cytotoxic agent is attached by a disulfide bondto the 6-mercapto group, and the transport polymer is bound to thecysteine carbonyl moiety via an amide linkage. Cleavage of the disulfidebond by reduction or disulfide exchange results in release of the freecytotoxic agent.

A method for synthesizing a disulfide-containing conjugate is providedin Example 9A. The product contains a heptamer of Arg residues which islinked to 6-mercaptopurine by an N-acetyl-Cys-Ala-Ala linker, where theAla residues are include as an additional spacer to render the disulfidemore accessible to thiols and reducing agents for cleavage within acell. The linker in this example also illustrates the use of amidebonds, which can be cleaved enzymatically within a cell.

In another approach, the conjugate includes a photocleavable linkerwhich is cleaved upon exposure to electromagnetic radiation. Anexemplary linkage is illustrated in FIG. 6B, which shows a conjugate(II) containing a transport polymer T which is linked to6-mercaptopurine via a meta-nitrobenzoate linking moiety. Polymer T islinked to the nitrobenzoate moiety by an amide linkage to the benzoatecarbonyl group, and the cytotoxic agent is bound via its 6-mercaptogroup to the p-methylene group. The compound can be formed by reacting6-mercaptopurine with p-bromomethyl-m-nitrobenzoic acid in the presnceof NaOCH₃/methanol with heating, followed by coupling of the benzoatecarboxylic acid to a transport polymer, such as the amino group of aγ-aminobutyric acid linker attached to the polymer (Example 9B).Photo-illumination of the conjugate causes release of the6-mercaptopurine by virtue of the nitro group that is ortho to themercaptomethyl moiety. This approach finds utility in phototherapymethods as are known in the art, particularly for localizing drugactivation to a selected area of the body.

Preferably, the cleavable linker contains first and second cleavablegroups that can cooperate to cleave the polymer from the biologicallyactive agent, as illustrated by the following approaches. That is, thecleavable linker contains a first cleavable group that is distal to theagent, and a second cleavable group that is proximal to the agent, suchthat cleavage of the first cleavable group yields a linker-agentconjugate containing a nucleophilic moiety capable of reactingintramolecularly to cleave the second cleavable group, thereby releasingthe agent from the linker and polymer.

FIG. 6C shows a conjugate (III) containing a transport polymer T linkedto the anticancer agent, 5-fluorouracil (5FU). Here, the linkage isprovided by a modified lysyl residue. The transport polymer is linked tothe α-amino group, and the 5-fluorouracil is linked via the α-carbonyl.The lysyl ε-amino group has been modified to a carbamate ester ofo-hydroxymethyl nitrobenzene, which comprises a first, photolabilecleavable group in the conjugate. Photoillumination severs thenitrobenzene moiety from the conjugate, leaving a carbamate which alsorapidly decomposes to give the free E-amino group, an effectivenucleophile. Intramolecular reaction of the ε-amino group with the amidelinkage to the 5-fluorouracil group leads to cyclization with release ofthe 5-fluorouracil group.

FIG. 6D illustrates a conjugate (IV) containing a transport polymer Tlinked to 2′-oxygen of the anticancer agent, paclitaxel. The linkage isprovided by a linking moiety that includes (i) a nitrogen atom attachedto the transport polymer, (ii) a phosphate monoester located para to thenitrogen atom, and (iii) a carboxymethyl group meta to the nitrogenatom, which is joined to the 2′-oxygen of paclitaxel by a carboxylateester linkage. Enzymatic cleavage of the phosphate group from theconjugate affords a free phenol hydroxyl group. This nucleophilic groupthen reacts intramolecularly with the carboxylate ester to release freepaclitaxel, for binding to its biological target. Example 9C describes asynthetic protocol for preparing this type of conjugate.

FIG. 6E illustrates yet another approach wherein a transport polymer islinked to a biologically active agent, e.g., paclitaxel, by anaminoalkyl carboxylic acid. Preferably, the linker amino group is linkedto the linker carboxyl carbon by from 3 to 5 chain atoms (n=3 to 5),preferably either 3 or 4 chain atoms, which are preferably provided asmethylene carbons. As seen in FIG. 6E, the linker amino group is joinedto the transport polymer by an amide linkage, and is joined to thepaclitaxel moiety by an ester linkage. Enzymatic cleavage of the amidelinkage releases the polymer and produces a free nucleophilic aminogroup. The free amino group can then react intramolecularly with theester group to release the linker from the paclitaxel.

FIGS. 6D and 6E are illustrative of another aspect of the invention,comprising taxane- and taxoid anticancer conjugates which have enhancedtrans-membrane transport rates relative to corresponding non-conjugatedforms. The conjugates are particularly useful for inhibiting growth ofcancer cells. Taxanes and taxoids are believed to manifest theiranticancer effects by promoting polymerization of microtubules (andinhibiting depolymerization) to an extent that is deleterious to cellfunction, inhibiting cell replication and ultimately leading to celldeath.

The term “taxane” refers to paclitaxel (FIG. 6F, R′=acetyl, R″=benzyl)also known under the trademark “TAXOL”) and naturally occurring,synthetic, or bioengineered analogs having a backbone core that containsthe A, B, C and D rings of paclitaxel, as illustrated in FIG. 6G. FIG.6F also indicates the structure of “TAXOTERE™” (R′=H, R″=BOC), which isa somewhat more soluble synthetic analog of paclitaxel sold byRhone-Poulenc. “Taxoid” refers to naturally occurring, synthetic orbioengineered analogs of paclitaxel that contain the basic A, B and Crings of paclitaxel, as shown in FIG. 6H. Substantial synthetic andbiological information is available on syntheses and activities of avariety of taxane and taxoid compounds, as reviewed in Suffness (1995),particularly in Chapters 12 to 14, as well as in the subsequentpaclitaxel literature. Furthermore, a host of cell lines are availablefor predicting anticancer activities of these compounds against certaincancer types, as described, for example, in Suffness at Chapters 8 and13.

The tranport polymer is conjugated to the taxane or taxoid moiety viaany suitable site of attachment in the taxane or taxoid. Conveniently,the transport polymer is linked via a C2′-oxygen atom, C7-oxygen atomor, using linking strategies as above. Conjugation of a transportpolymer via a C7-oxygen leads to taxane conjugates that have anticancerand antitumor activity despite conjugation at that position.Accordingly, the linker can be cleavable or non-cleavable. Conjugationvia the C2′-oxygen significantly reduces anticancer activity, so that acleavable linker is preferred for conjugation to this site. Other sitesof attachment can also be used, such as C10.

It will be appreciated that the taxane and taxoid conjugates of theinvention have improved water solubility relative to taxol (∞0.25 μg/mL)and taxotere (6-7 μg/mL). Therefore, large amounts of solubilizingagents such as “CREMOPHOR EL” (polyoxyethylated castor oil), polysorbate80 (polyoxyethylene sorbitan monooleate, also known as “TWEEN 80”), andethanol are not required, so that side-effects typically associated withthese solubilizing agents, such as anaphylaxis, dyspnea, hypotension,and flushing, can be reduced.

B. Metals

Metals can be transported into eukaryotic and prokaryotic cells usingchelating agents such as texaphyrin or diethylene triamine pentaceticacid (DTPA), conjugated to a transport membrane of the invention, asillustrated by Example 10. These conjugates are useful for deliveringmetal ions for imaging or therapy. Exemplary metal ions include Eu, Lu,Pr, Gd, Tc99m, Ga67, In111, Y90, Cu67, and Co57. Preliminarymembrane-transport studies with conjugate candidates can be performedusing cell-based assays such as described in the Example section below.For example, using europium ions, cellular uptake can be monitored bytime-resolved fluorescence measurements. For metal ions that arecytotoxic, uptake can be monitored by cytotoxicity.

C. Macromolecules

The enhanced transport method of the invention is particularly suitedfor enhancing transport across biological membranes for a number ofmacromolecules, including, but not limited to proteins, nucleic acids,polysaccharides, and analogs thereof. Examplary nucleic acids includeoligonucleotides and polynucleotides formed of DNA and RNA, and analogsthereof, which have selected sequences designed for hybridization tocomplementary targets (e.g., antisense sequences for single- ordouble-stranded targets), or for expressing nucleic acid transcripts orproteins encoded by the sequences. Analogs include charged andpreferably uncharged backbone analogs, such as phosphonates (preferablymethyl phosphonates), phosphoramidates (N3′ or N5′), thiophosphates,uncharged morpholino-based polymers, and protein nucleic acids (PNAs).Such molecules can be used in a variety of therapeutic regimens,including enzyme replacement therapy, gene therapy, and anti-sensetherapy, for example.

By way of example, protein nucleic acids (PNA) are analogs of DNA inwhich the backbone is structurally homomorphous with a deoxyribosebackbone. It consists of N-(2-aminoethyl)glycine units to which thenucleobases are attached. PNAs containing all four natural nucleobaseshybridize to complementary oligonucleotides obeying Watson-Crickbase-pairing rules, and is a true DNA mimic in terms of base pairrecognition (Egholm et al., 1993). The backbone of a PNA is formed bypeptide bonds rather than phosphate esters, making it well-suited foranti-sense applications. Since the backbone is uncharged, PNA/DNA orPNA/RNA duplexes that form exhibit greater than normal thermalstability. PNAs have the additional advantage that they are notrecognized by nucleases or proteases. In addition, PNAs can besynthesized on an automated peptides synthesizer using standard t-Bocchemistry. The PNA is then readily linked to a transport polymer of theinvention.

Examples of anti-sense oligonucleotides whose transport into cells maybe enhanced using the methods of the invention are described, forexample, in U.S. Pat. No. 5,594,122. Such oligonucleotides are targetedto treat human immunodeficiency virus (HIV). Conjugation of a transportpolymer to an anti-sense oligonucleotide can be effected, for example,by forming an amide linkage between the peptide and the 5′-terminus ofthe oligonucleotide through a succinate linker, according towell-established methods. The use of PNA conjugates is furtherillustrated in Example 11.

FIG. 7 shows results obtained with a conjugate of the inventioncontaining a PNA sequence for inhibiting secretion of gamma-interferon(γ-IFN) by T cells, as detailed in Example 11. As can be seen, theanti-sense PNA conjugate was effective to block γ-IFN secretion when theconjugate was present at levels above about 10 μM. In contrast, noinhbition was seen with the sense-PNA conjugate or the non-conjugatedantisense PNA alone.

Another class of macromolecules that can be transported acrossbiological membranes is exemplified by proteins, and in particular,enzymes. Therapeutic proteins include, but are not limited toreplacement enzymes. Therapeutic enzymes include, but are not limitedto, alglucerase, for use in treating lysozomal glucocerebrosidasedeficiency (Gaucher's disease), alpha-L-iduronidase, for use in treatingmucopolysaccharidosis I, alpha-N-acetylglucosamidase, for use intreating sanfilippo B syndrome, lipase, for use in treating pancreaticinsufficiency, adenosine deaminase, for use in treating severe combinedimmunodeficiency syndrome, and triose phosphate isomerase, for use intreating neuromuscular dysfunction associated with triose phosphateisomerase deficiency.

In addition, and according to an important aspect of the invention,protein antigens may be delivered to the cytosolic compartment ofantigen-presenting cells (APCs), where they are degraded into peptides.The peptides are then transported into the endoplasmic reticulum, wherethey associate with nascent HLA class I molecules and are displayed onthe cell surface. Such “activated” APCs can serve as inducers of class Irestricted antigen-specific cytotoxic T-lymphocytes (CTLs), which thenproceed to recognize and destroy cells displaying the particularantigen. APCs that are able to carry out this process include, but arenot limited to, certain macrophages, B cells and dendritic cells. In oneembodiment, the protein antigen is a tumor antigen for eliciting orpromoting an immune response against tumor cells.

The transport of isolated or soluble proteins into the cytosol of APCwith subsequent activation of CTL is exceptional, since, with fewexceptions, injection of isolated or soluble proteins does not resulteither in activation of APC or induction of CTLs. Thus, antigens thatare conjugated to the transport enhancing compositions of the presentinvention may serve to stimulate a cellular immune response in vitro orin vivo.

Example 14 provides details of experiments carried out in support of thepresent invention in which an exemplary protein antigen, ovalbumin, wasdelivered to APCs after conjugation to an R7 polymer. Subsequentaddition of the APCs to cytotoxic T lymphocytes (CTLs) resulted in CD8+albumin-specific cytotoxic T cells (stimulated CTLs). In contrast, APCsthat had been exposed to unmodified ovalbumin failed to stimulate theCTLs.

In parallel experiments, histocompatible dendritic cells (a specifictype of APC) were exposed to ovalbumin-R7 conjugates, then injected intomice. Subsequent analysis of blood from these mice revealed the presenceof albumin-specific CTLs. Control mice were given dendritic cells thathad been exposed to unmodified albumin. The control mice did not exhibitthe albumin-specific CTL response. These experiments exemplify one ofthe specific utilities associated with delivery of macromolecules ingeneral, and proteins in particular, into cells.

In another embodiment, the invention is useful for deliveringimmunospecific antibodies or antibody fragments to the cytosol tointerfere with deleterious biological processes such as microbialinfection. Recent experiments have shown that intracellular antibodiescan be effective antiviral agents in plant and mammalian cells (e.g.,Tavladoraki et al., 1993; and Shaheen et al., 1996). These methods havetypically used single-chain variable region fragments (scFv), in whichthe antibody heavy and light chains are synthesized as a singlepolypeptide. The variable heavy and light chains are usually separatedby a flexible linker peptide (e.g., of 15 amino acids) to yield a 28 kDamolecule that retains the high affinity ligand binding site. Theprincipal obstacle to wide application of this technology has beenefficiency of uptake into infected cells. But by attaching transportpolymers to scFv fragments, the degree of cellular uptake can beincreased, allowing the immunospecific fragments to bind and disableimportant microbial components, such as HIV Rev, HIV reversetranscriptase, and integrase proteins.

D. Peptides

Peptides to be delivered by the enhanced transport methods describedherein include, but should not be limited to, effector polypeptides,receptor fragments, and the like. Examples include peptides havingphosphorylation sites used by proteins mediating intracellular signals.Examples of such proteins include, but are not limited to, proteinkinase C, RAF-1, p21Ras, NF-κB, C-JUN, and cytoplasmic tails of membranereceptors such as IL-4 receptor, CD28, CTLA-4, V7, and MHC Class I andClass II antigens.

When the transport enhancing molecule is also a peptide, synthesis canbe achieved either using an automated peptide synthesizer or byrecombinant methods in which a polynucleotide encoding a fusion peptideis produced, as mentioned above.

In experiments carried out in support of the present invention (Example15) a 10-amino acid segment of the cytoplasmic tail region of thetransmembrane protein V7 (also known as CD101) was synthesized with anR7 polymer sequence at its C terminus. This tail region is known tophysically associate with and mediate the inactivation of RAF-1 kinase,a critical enzyme in the MAP kinase pathway of cellular activation. TheV7-R7 conjugate was added to T-cells, which were subsequently lysed withdetergent. The soluble fraction was tested for immunoprecipitation byanti-V7 murine antibody in conjunction with goat anti-mouse IgG.

In the absence of peptide treatment, RAF-1, a kinase known to associatewith and be inactivated by association with V7, co-precipitated with V7.In peptide treated cells, RAF-1 protein was eliminated from the V7immuno-complex. The same peptide treatment did not disrupt a complexconsisting of RAF-1 and p21 Ras, ruling out any non-specificmodification of RAF-1 by the V7 peptides. These results showed that acytoplasmic tail region V7 peptide, when conjugated to a membranetransport enhancing peptide of the present invention, enters a targetcell and specifically associates with a physiological effector molecule,RAF-1. Such association can be used to disrupt intracellular processes.

In a second set of studies, the V7 portion of the conjugate wasphosphorylated in vitro using protein kinase C. Anti-RAF-1 precipitatesof T cells that had been exposed to the phosphorylated V7 tail peptides,but not the unphosphorylated V7 tail peptide, demonstrated potentinhibition of RAF-kinase activity. These studies demonstrate twoprinciples. First, the transport polymers of the invvention can effecttransport of a highly charged (phosphorylated) molecule across the cellmembrane. Second, while both phosphorylated and unphosphorylated V7 tailpeptides can bind to RAF-1, only the phosphorylated peptide modifiedRAF-1 kinase activity.

VI. Screening Assay Method and Library

In another embodiment, the invention can be used to screen one or moreconjugates for a selected biological activity, wherein the conjugate(s)are formed from one or more candidate agents. Conjugate(s) are contactedwith a cell that exhibits a detectable signal upon uptake of theconjugate into the cell, such that the magnitude of the signal isindicative of the efficacy of the conjugate with respect to the selectedbiological activity.

One advantage of this embodiment is that it is particularly useful fortesting the activities of agents that by themselves are unable, orpoorly able, to enter cells to manifest biological activity. Thus, theinvention provides a particularly efficient way of identifying activeagents that might not otherwise be accessible through large-scalescreening programs, for lack of an effective and convenient way oftransporting the agents into the cell or organelle.

Preferably, the one or more candidate agents are provided as acombinatorial library of conjugates which are prepared using any of anumber of combinatorial synthetic methods known in the art. For example,Thompson and Ellman (1996) recognized at least five different generalapproaches for preparing combinatorial libraries on solid supports,namely (1) synthesis of discrete compounds, (2) split synthesis (splitand pool), (3) soluble library deconvolution, (4) structuraldetermination by analytical methods, and (5) encoding strategies inwhich the chemical compositions of active candidates are determined byunique labels, after testing positive for biological activity in theassay. Synthesis of libraries in solution includes at least (1)spatially separate syntheses and (2) synthesis of pools (Thompson,supra). Further description of combinatorial synthetic methods can befound in Lam et al. (1997), which particularly describes theone-bead-one-compound approach.

These approaches are readily adapted to prepare conjugates in accordancewith the present invention, including suitable protection schemes asnecessary. For example, for a library that is constructed on one or moresolid supports, a transport peptide moiety can be attached to thesupport(s) first, followed by building or appending candidate agentscombinatorially onto the polymers via suitable reactive functionalities.In an alternative example, a combinatorial library of agents is firstformed on one or more solid supports, followed by appending a transportpolymer to each immobilized candidate agent. Similar or differentapproaches can be used for solution phase syntheses. Libraries formed ona solid support are preferably severed from the support via a cleavablelinking group by known methods (Thompson et al., and Lam et al., supra).

The one or more conjugate candidates can be tested with any of a numberof cell-based assays that elicit detectable signals in proportion to theefficacy of the conjugate. Conveniently, the candidates are incubatedwith cells in multiwell plates, and the biological effects are measuredvia a signal (e.g., fluorescence, reflectance, absortpion, orchemiluminescence) that can be quantitated using a plate reader.Alternatively, the incubation mixtures can be removed from the wells forfurther processing and/or analysis. The structures of active andoptionally inactive compounds, if not already known, are thendetermined, and this information can be used to identify lead compoundsand to focus further synthesis and screening efforts.

For example, the γ-interferon secretion assay detailed in Example 11 isreadily adapted to a multiwell format, such that active secretioninhibitors can be detected by europium-based fluorescence detectionusing a plate reader. Anticancer agents can be screened usingestablished cancer cell lines (e.g., provided by the National Institutesof Health (NIH) and the National Cancer Institute (NCI). Cytotoxiceffects of anticancer agents can be determined by trypan dye exclusion,for example.

Other examples include assays directed to inhibiting cell signaling,such as IL-4 receptor inhibition; assays for blocking cellularproliferation, and gene expression assays. In a typical gene expressionassay, a gene of interest is placed under the control of a suitablepromotor and is followed downstream by a gene for producing a reporterspecies such as β-galactosidase or firefly luciferase. An inhibitoryeffect can be detected based on a decrease in reporter signal.

The invention also includes a conjugate library which is useful forscreening in the above method. The library includes a plurality ofcandidate agents for one or more selected biological activities, each ofwhich is conjugated to at least one transport polymer in accordance withthe invention. Preferably, the conjugate library is a combinatoriallibrary. In another embodiment, the invention includes a regular arrayof distinct polymer-agent conjugates distributed in an indexed orindexable plurality of sample wells, for testing and identifying activeagents of interest.

VI. Utility

Compositions and methods of the present invention have particularutility in the area of human and veterinary therapeutics. Generally,administered dosages will be effective to deliver picomolar tomicromolar concentrations of the therapeutic composition to the effectorsite. Appropriate dosages and concentrations will depend on factors suchas the therapeutic composition or drug, the site of intended delivery,and the route of administration, all of which can be derived empiricallyaccording to methods well known in the art. Further guidance can beobtained from studies using experimental animal models for evaluatingdosage, as are known in the art.

Administration of the compounds of the invention with a suitablepharmaceutical excipient as necessary can be carried out via any of theaccepted modes of administration. Thus, administration can be, forexample, intravenous, topical, subcutaneous, transcutaneous,intramuscular, oral, intra-joint, perenteral, peritoneal, intranasal, orby inhalation. The formulations may take the form of solid, semi-solid,lyophilized powder, or liquid dosage forms, such as, for example,tablets, pills, capsules, powders, solutions, suspensions, emulsions,suppositories, retention enemas, creams, ointments, lotions, aerosols orthe like, preferably in unit dosage forms suitable for simpleadministration of precise dosages.

The compositions typically include a conventional pharmaceutical carrieror excipient and may additionally include other medicinal agents,carriers, adjuvants, and the like. Preferably, the composition will beabout 5% to 75% by weight of a compound or compounds of the invention,with the remainder consisting of suitable pharmaceutical excipients.Appropriate excipients can be tailored to the particular composition androute of administration by methods well known in the art, e.g.,(Gennaro, 1990).

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. The composition may take the form of a solution, suspension,tablet, pill, capsule, powder, sustained-release formulation, and thelike.

In some embodiments, the pharmaceutical compositions take the form of apill, tablet or capsule, and thus, the composition can contain, alongwith the biologically active conjugate, any of the following: a diluentsuch as lactose, sucrose, dicalcium phosphate, and the like; adisintegrant such as starch or derivatives thereof; a lubricant such asmagnesium stearate and the like; and a binder such a starch, gum acacia,polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

The active compounds of the formulas may be formulated into asuppository comprising, for example, about 0.5% to about 50% of acompound of the invention, disposed in a polyethylene glycol (PEG)carrier (e.g., PEG 1000 [96%] and PEG 4000 [4%]).

Liquid compositions can be prepared by dissolving or dispersing compound(about 0.5% to about 20%), and optional pharmaceutical adjuvants in acarrier, such as, for example, aqueous saline (e.g., 0.9% w/v sodiumchloride), aqueous dextrose, glycerol, ethanol and the like, to form asolution or suspension, e.g., for intravenous administration. The activecompounds may also be formulated into a retention enema.

If desired, the composition to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, such as, for example, sodium acetate,sorbitan monolaurate, or triethanolamine oleate.

For topical administration, the composition is administered in anysuitable format, such as a lotion or a transdermal patch. For deliveryby inhalation, the composition can be delivered as a dry powder (e.g.,Inhale Therapeutics) or in liquid form via a nebulizer.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (1980). The composition to be administered will, in any event,contain a quantity of the pro-drug and/or active compound(s) in apharmaceutically effective amount for relief of the condition beingtreated when administered in accordance with the teachings of thisinvention.

Generally, the compounds of the invention are administered in atherapeutically effective amount, i.e., a dosage sufficient to effecttreatment, which will vary depending on the individual and conditionbeing treated. Typically, a therapeutically effective daily dose is from0.1 to 100 mg/kg of body weight per day of drug. Most conditions respondto administration of a total dosage of between about 1 and about 30mg/kg of body weight per day, or between about 70 mg and 2100 mg per dayfor a 70 kg person.

Stability of the conjugate can be further controlled by the compositionand stereochemistry of the backbone and sidechains of the polymer. Forpolypeptide polymers, D-isomers are generally resistant to endogenousproteases, and therefore have longer half-lives in serum and withincells. D-polypeptide polymers are therefore appropriate when longerduration of action is desired. L-polypeptide polymers have shorterhalf-lives due to their susceptibility to proteases, and are thereforechosen to impart shorter acting effects. This allows side-effects to beaverted more readily by withdrawing therapy as soon as side-effects areobserved. Polypeptides comprising mixtures of D and L-residues haveintermediate stabilities. Homo-D-polymers are generally preferred.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE 1 Peptide Synthesis

Peptides were synthesized using solid phase techniques on an AppliedBiosystems Peptide synthesizer using FastMOC™ chemistry and commerciallyavailable Wang resins and Fmoc protected amino acids, according tomethods well known in the art (Bonifaci). Peptides were purified usingC4 or C18 reverse phase HPLC columns, and their structures wereconfirmed using amino acid analysis and mass spectrometry.

EXAMPLE 2 Fluorescence Assays

Fluorescent peptides were synthesized by modification of the aminoterminus of the peptide with aminocaproic acid followed by reaction withfluorescein isothiocyanate in the presence of(2-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate/N-hydroxy benzotriazole dissolved in N-methylpyrrolidone. The products were purified by gel filtration.

Suspension cells (10⁶/mL) were incubated for varying times, at 37° C.,23° C., or 4° C., with a range of concentrations of peptides orconjugates in PBS pH 7.2 containing 2% fetal calf serum (PBS/FCS) in 96well plates. After a 15 minute incubation, the cells were pelleted bycentrifugation, washed three times with PBS/FCS containing 1% sodiumazide, incubated with trypsin/EDTA (Gibco) at 37° C. for five minutes,then washed twice more with PBS/FCS/NaN₃. The pelleted cells wereresuspended in PBS containing 2% FCS and 0.1% propidium iodide andanalyzed on a FACScan (Becton Dickenson, Mountain View, Calif.). Cellspositive for propidium iodide were excluded from the analysis. Foranalysis of polymers of arginine, the voltage of the photomultiplier wasreduced by an order of magnitude to allow a more accurate measurement.

EXAMPLE 3 Tat Basic Peptide Ver sus Poly-Arg Peptides

Uptake levels of the following polypeptides were measured by the methodin Example 2: (1) a polypeptide comprising HIV tat residues 49-57(Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg=SEQ ID NO:1), (2) a nonamer ofL-Lys residues (K9, SEQ ID NO:2), and (3) homo-L or homo-D polypeptidescontaining four to nine Arg residues (SEQ ID NO:3-8 and 12-17). Resultsa re shown in FIGS. 2A-2C.

EXAMPLE 4 Confocal Cell Microscopy

cells incubated with fluorescent polyarginine peptides were prepared asdescribed above for binding assays and analyzed at the Cell SciencesImaging Facility (Stanford University, Stanford, Calif.) using ascanning, single beam laser confocal microscope, with an excitationwavelength of 488 nm (argon-ion laser) and an emission band-width of510-550 using a band-pass filter. Conjugates (6.25 μM) containingtat(49-57), R7, or r7 coupled to fluorescein were incubated with Jurkatcells for 37° C. for 10 minutes. FIGS. 2A-2F show result s for emittedfluorescence (FIGS. 2A-2C) and transmitted light (2D-2F) for tat(49-57)(FIGS. 2A and 2C), R7 (FIGS. 2B and 2E), and r7 (FIGS. 2C and 2F).

EXAMPLE 5 Length Range Studies

The following homopolymers of polyarginine were tested by thefluorescence assay in Example 2, with incubation at 37° C. for 15minutes prior to cell pelleting: r9, R9, R15, R20, R25, and R30. Inaddition, a mixture of L-arginine polymers having an average molecularweight of 12,000 daltons (approximately 100 amino acids) was also tested(Sigma Chem. Co.) after being labeled with fluorescein isothiocyanateand purified by gel filtration (“SEPHADEX” G-25). The cells wereanalyzed by FACS, and the mean fluorescence of the live cells wasmeasured. Cytotoxicity of each conjugate was also measured bycalculating the percentage of cells that stained with propidium iodide,which is characteristic of cell death. Uptake results for the r9, R9,R15, R20, and R25 conjugates are shown in FIG. 3.

The commercially available polyarginine (12,000 MW) precipitatedproteins in serum, most likely α1-acid glycoprotein. Therefore, thelevel of fetal calf serum was reduced 10-fold in the assay forconjugates prepared from this material.

The 12,000 MW poly-Arg composition was toxic at concentrations from 800nM to 50 μM and is excluded from FIG. 3. Poly-L-Arg conjugatescontaining 20 arginine residues or more were toxic at concentrationsgreater than 12 μM, such that toxicity increased with length.

EXAMPLE 6 Kinetics of Uptake

To measure Vmax and Km parameters of cellular uptake, the assay methodof Example 2 was used with the following modifications. Peptides wereincubated with cells for 0.5, 1, 2, and 4 minutes at 4° C. intriplicate, in 50 μL of PBS/FCS in 96-well plates. At the end ofincubation, the reaction was quenched by diluting the samples in 5 mL ofPBS/FCS, centrifuging and washing once with PBS/FCS, trypsin/EDTA, andfinally again with PBS/FCS, and taking up the pellets in PBS/FCScontaining propidium iodide for analysis on a FACScan. FACS data werefitted to the Line-weaver-Burk equation for Michaelis-Menten kinetics.Kinetic data for fluorescent conjugates of tat(49-57), R9, and r9 areshown in Table 1 above.

EXAMPLE 7 Metabolic Inhibitor Effects on Transport

Suspension cells (10⁶/mL) were incubated for 30 minutes with 0.5% sodiumazide in PBS containing 2% FCS. At the end of incubation, fluorescentpeptides (tat(49-57)), R7, R8, or R9) were added to a finalconcentration of 12.5 μM. After incubation for 30 minutes, the cellswere washed as in Example 2, except that all wash buffers contained 0.1%sodium azide. The results are shown in FIG. 4.

EXAMPLE 8 Transport into Bacterial Cells

Gram-negative bacteria (E. coli strain HB101) and gram-positive bacteria(Strep. bovis) were grown in appropriate media in logarithmic phase.Cell cultures (4×108 per mL) were incubated for 30 minutes at 37° C.with varying concentrations of fluorescent conjugates containing linearpolymers of L-arginine (R4 through R9), D-arginine (r4 through r9), orL-lysine (K9) at conjugate concentrations of 3 to 50 μM. The cells werewashed and taken up in PBS-containing propidium iodide (to distinguishdead cells) and analyzed by FACS and fluorescent microscopy. Results areshown in FIGS. 5A-5C as discussed above.

EXAMPLE 9 Conjugates with Exemplary Cleavable Linkers

A. 6-Mercaptopurine Cysteine Dissulfide Conjugate

A1. Thiol Activation. N-acetyl-Cys(SH)-Ala-Ala-(Arg)7-CO₂H (12.2 mg,0.0083 mmol) was dissolved in 3 mL of 3:1 AcOH:H₂O with stirring atambient temperature. To this solution was added dithio-bis(5-nitropyridine) (DTNP) (12.9 mg, 0.0415 mmol, 5 eq). The solution waspermitted to stir for 24 h at ambient temperature, after which themixture took on a bright yellow color. Solvent was removed in vacuo, andthe residue was redissolved in 5 mL of H₂O and extracted 3 times withethyl acetate to remove excess DTNP. The aqueous layer was lyophilized,and the product was used without further purification.

A2. Attachment of Drug N-acetyl-Cys(SH)-Ala-Ala-(Arg)7-CO₂H (0.0083mmol) was dissolved in 1 mL of degassed H₂O (pH=5) under argon at roomtemperature, with stirring. 6-Mercaptopurine (1.42 mg, 0.0083 mmol, 1eq) in 0.5 mL DMF was added to the mixture. The reaction was permittedto stir for 18 h under inert atmosphere at ambient temperature. After 18h, a bright yellow color devloped, indicating the presence of free5-nitro-2-thiopyridine. Solvent was removed under reduced pressure, andthe residue was purified by HPLC, providing the desired product (I, FIG.6A) in 50% overall yield.

B. Photocleavable Taxol Conjugate

3-Nitro-4-(bromomethyl)benzoic acid (100 mg, 0.384 mmol) is dissolved inanhydrous methanol (5 mL) under an atmosphere of nitrogen. To thissoluition is added sodium methoxide (88 μL, 25% (w/w) in methanol, 0.384mmol, 1 eq) followed by addition of 6-mercaptopurine (58.2 mg, 0.384mmol, 1 eq). The mixture is warmed to reflux and permitted to stir for 3h. The reaction mixture is then cooled, filtered, and quenched byacidification with 6N HCl. The reaction volume is then reduced toone-half at which point the product precipitates and is collected byfiltration. The residue is redissolved in methanol, filtered (ifnecessary) and concentrated under reduced pressure to provide desiredsulfide (II, FIG. 6B) in 50% yield as a yellow powdery solid.

C. Phosphate-Cleavable Taxol Conjugate

C1. To a suspension of o-hydroxy phenylacetic acid (15.0 g, 0.099 mol)in H₂O (39 mL) at 0° C. was added a solution of nitric acid (12 mL of65% in 8 mL H₂O) slowly via pipette. The solution was stirred for anadditional 1.5 h at 0° C. The mixture was then warmed to ambienttemperature and allowed to stir for an additional 0.5 h. Theheterogenous solution was poured over ice (10 g) and filtered to removethe insoluble ortho-nitro isomer. The reddish solution was concentratedunder reduced pressure, and the thick residue was redissolved in 6N HCland filtered through celite. The solvent was again removed under reducedpressure to provide the desired 2-hydroxy-4-nitro-phenylacetic acid as alight, brownish-red solid (40% yield). The product (IV-a) was used inthe next step without further purification.

C2. Product IV-a (765 mg, 3.88 mmol) was dissolved in freshly distilledTHF (5 mL) under argon atmosphere. The solution was cooled to 0° C., andborane-THF (1.0 M in THF, 9.7 mL, 9.7 mmol, 2.5 eq) was added dropwisevia syringe with apparent evolution of hydrogen. The reaction waspermitted to stir for an additonal 16 h, slowly warming to roomtemperature. The reaction was quenched by slow addition of 1M HCl (withfurious bubbling) and 10 mL of ethyl acetate. The layers were separatedand the aqueous layer extracted five times with ethyl acetate. Thecombined organic layers were washed with brine and dried over magnesiumsulfate. The solvent was evaporated in vacuo and the residue purified byrapid column chromatography (1:1 hexane:ethyl acetate) to provide thedesired nitro-alcohol (IV-b) as a light yellow solid (85w yield).

C3. Nitro-alcohol (IV-b) (150 mg, 0.819 mmol) was dissolved in dry DMF(5 mL) containing di-t-butyldi-carbonate (190 mg, 1.05 eq) and 10% Pd-C(10 mg). The mixture was placed in a Parr apparatus andpressurized/purged five times. The solution was then pressurized to 47psi and allowed to shake for 24 h. The reaction was quenched byfiltration through celite, and the solvent was removed under reducedpressure. The residue was purified by column chromatography (1:1hexane:ethyl acetate) to provide the protected aniline product (IV-c) asa tan crystalline solid in 70% yield.

C4. TBDMS-Cl (48 mg, 0.316 mmol) was dissolved in freshly distilleddichloromethane (4 mL) under an argon atmosphere. To this solution wasadded imidazole (24 mg, 0.347 mmol, 1.1 eq) and immediately a whiteprecipitate formed. The solution was stirred for 30 min at roomtemperature, at which point product IV-c (80 mg, 0.316 mmol, 1.0 eq) wasadded rapidly as a solution in dichloromethane/THF (1.0 mL). Theresulting mixture was permitted to stir for an additional 18 h atambient temperature. Reaction was quenched by addition of saturatedaqueous ammonium chloride. The layers were separated and the aqueousphase extracted 3 times with ethyl acetate and the combined organiclayers washed with brine and dried over sodium sulfate. The organicphase was concentrated to provide silyl ether-phenol product (IV-d) as alight yellow oil (90% yield).

C5. Silyl ether-phenol IV-d (150 mg, 0.408 mmol) was dissolved infreshly distilled THF (7 mL) under argon and the solution cooled to 0°C. n-BuLi (2.3 M in hexane, 214 uL) was then added dropwise via syringe.A color change from light yellow to deep red was noticed immediately.After 5 min, tetrabenzyl pyrophosphate (242 mg, 0.45 mmol, 1.1 eq) wasadded rapidly to the stirring solution under argon. The solution wasstirred for an additional 18 h under inert atmosphere, slowly warming toroom temperature, during which time a white precipitate forms. Thereaction was quenched by addition of saturated aqueous ammonium chlorideand 10 mL of ethyl acetate. The layers were separated, and the aqueouslayer was extracted 5 times with ethyl acetate. The combined organicphases were washed with brine and dried over magnesium sulfate. Thesolvent was removed by evaporation and the residue purified by rapidcolumn chromatography (1:1 hexane:ethyl acetate) to provide the desiredphosphate-silyl ether (IV-e) as a light orange oil (90% yield).

C6. Phosphate-silyl ether (IV-e) (10 mg, 0.0159 mmol) was dissolved in 2mL of dry ethanol at room temperature. To the stirring solution wasadded 20 uL of conc. HCl (1% v:v solution), and the mixture waspermitted to stir until TLC analysis indicated the reaction wascomplete. Solid potassium carbonate was added to quench the reaction,and the mixture was rapidly filtered through silica gel and concentratedto give crude alcohol-dibenzyl phosphate product (IV-f) as a lightyellow oil (100% yield).

C7. Alcohol IV-f (78 mg, 0.152 mmol) was dissolved in freshly distilleddichloromethane (10 mL) under an argon atmosphere. To the solution wasadded Dess-Martin periodinane (90 mg, 0.213 mmol, 1.4 eq). The solutionwas permitted to stir, and the progress of the reaction was monitored byTLC analysis. Once TLC indicated completion, reaction was quenched byaddition of 1:1 saturated aqueous sodium bicarbonate:saturated aqueoussodium thiosulfite. The biphasic mixture was permitted to stir for 1 hat ambient temperature. The layers were separated, and the aqueous phasewas extracted 3 times with ethyl acetate. The combined oragnic layerswere washed with brine and dried over sodium sulfate. Solvent wasremoved under reduced pressure to provide aldehyde product (IV-g) as alight tan oil (100% yield).

C8. Aldehyde IV-g (78 mg, 0.152 mmol) was dissolved in t-butanol/water(3.5 mL) under inert atmosphere. To the rapidly stirring solution wasadded 2-methyl-2-butene (1.0 M in THF, 1.5 mL), sodiumphosphate-monobasic (105 mg, 0.76 mmol, 5 eq) and sodium chlorite (69mg, 0.76 mmol, 5 eq). The solution was permitted to stir for 8additional hours at room temperature. The solution was concentrated, andthe residue was acidified and extracted with ethyl acetate 3 times. Thecombined organic phases were dried over magnesium sulfate. The solutionwas again concentrated under reduced pressure and the residue waspurified via column chromatography (2:1 ethyl acetate:hexane) to givethe desired carboxylic acid-dibenzylphosphate (IV-h) as a light yellowoil (65% yield).

C9. Acid IV-h (8.0 mg, 0.0152 mmol, 1.1 eq) was dissolved in freshlydistilled dichloromethane (2 mL) under argon at ambient temperature. Tothis mixture was added paclitaxel (12 mg, 0.0138 mmol, 1 eq) followed byDMAP (2 mg, 0.0138 mmol, 1 eq) and DCC (3.2 mg, 0.0152, 1.1 eq). Themixture was allowed to stir at room temperature for an additional 4 h,during which a light precipitate formed. Once TLC analysis indicatedthat the reaction was complete, solvent was removed under reducedpressure, and the residue was purified by rapid column chromatography(1:1 hexane:ethyl acetate) to provide paclitaxel-C2′-carboxylate ester(IV-i) as a white, crystalline solid (65% yield).

C10. Ester IV-i (5.0 mg) was dissolved in neat formic acid (1.0 mL)under an argon atmosphere at room temperature and permitted to stir for30 min. Once TLC indicated that the reaction was complete, the solutionwas concentrated under reduced pressure and the residue purified byrapid filtration through silica gel to give the desired anilinetaxolcompound (IV-j) in 50% yield as a white powder.

C11. To a solution of (poly di-CBZ)-protected AcHN-RRRRRRR-CO2H (1.2 eq,0.1 to 1.0 M) in dry DMF was added O-benzotriazolyloxytetramethyluronium hexafluorophosphate (HATU, 1.0 eq) and a catalyticamount of DMAP (0.2 eq). The solution stirred under inert atmosphere for5 min at ambient temperature. To this mixture was then addedTaxol-aniline derivative (IV-j) as a soluition in dry DMF (minimalvolume to dissolve). The resulting solution was stirred for anadditional 5 h at room temperature. Reaction was terminated byconcentrating the reaction mixture under reduced pressure. The crudereaction mixture was then purified by HPLC to provide the desiredmaterial (IV, FIG. 6D).

EXAMPLE 10 Transport of Metal Ions

3.93 g of DTPA is dissoved in 100 mls of HEPES buffer and 1.52 ml ofeuropium chloride atomic standard solution (Aldrich) dissolved in 8 mlof HEPES buffer is added and stirred for 30 minutes at room temperature.Chromatographic separation and lyophilization affords an Eu-DTPA chelatecomplex. This complex is then conjugated to the amino terminus of apolypeptide by solid phase peptide chemistry. The cellular uptake ofeuropium ion can be monitored by time resolved fluorescence.

EXAMPLE 11 Uptake of PNA-Peptide Conjugates

PNA peptide conjugates were synthesized using solid phase chemistry withcommercially available Fmoc reagents (PerSeptive Biosystems, Cambridge,Mass.) on either an Applied Biosystems 433A peptide synthesizer or aMillipore Expedite nucleic acid synthesis system. Polymers of D orL-arginine were attached to the amino or carboxyl termini of the PNAs,which are analogous to the 5′ and 3′ ends of the nucleic acids,respectively. The conjugates were also modified to include fluoresceinor biotin by adding an aminocaproic acid spacer to the amino terminus ofthe conjugate and then attaching biotin or fluorescein. The PNA-peptideconjugates were cleaved from the solid phase resin using 95% TFA, 2.5%triisopropyl silane, and 2.5% aqueous phenol. The resin was removed byfiltration, and residual acid was removed by evaporation. The productwas purified by HPLC using a C-18 reverse phase column, and the productwas lyophilized. The desired PNA-polymer conjugates were identifiedusing laser desorption mass spectrometry.

A. Inhibition of Cellular Secretion of Gamma-IFN

1. PNA-Peptide Conjugates. The following sense and antisense PNA-peptideconjugates were prepared for inhibiting gamma-IFN production, wherer=D-arginine, and R=L-arginine:

Sense:

NH₂-rrrrrrr-AACGCTACAC-COOH (SEQ ID NO:6 conjugated to SEQ ID NO:18)

Antisense:

NH₂-rrrrrrr-GTGTAGCGTT-COOH (SEQ ID NO:6 conjugated to SEQ ID NO:19)

Fluorescent antisense:

X-rrrrrrr-GTGTAGCGTT-COOH (X-SEQ ID NO:6 conjugated to SEQ ID NO:19)

where X=fluourescein-aminocaproate

Biotinylated antisense:

Z-rrrrrrr-GTGTAGCGTT-COOH (Z-SEQ ID NO:6 conjugated to SEQ ID NO:19)

where Z=biotin-aminocaproate

2. Uptake by T Cells. To show that PNA-polyarginine conjugates entercells effectively, the fluorescent antisense conjugate above (XSEQ IDNO:6 conjugated to SEQ ID NO:19) was synthesized by conjugatingfluorescein isothiocyanate to the amino terminus of r7 the aboveconjugated with SEQ ID NO:18 using an aminocaproic acid spacer.

Cellular uptake was assayed by incubating the Jurkat human T cell line(5×10⁵ cells/well) either pretreated for 30 minutes with 0.5% sodiumazide or phosphate buffered saline, with varying amounts (100 nM to 50μM) of the fluorescein-labeled sense and antisense PNA-r7 conjugate, aswell as the antisense PNA alone (without r7 segment). The amount ofantisense PNA that entered the cells was analyzed by confocal microscopyand FACS. In both cases, fluorescent signals were present only in cellsnot exposed to azide, and the fluorescent signal was dependent on thedose of the fluorescent conjugate and on the temperature and duration ofincubation.

3. Gamma-IFN Assay. The amount of gamma interferon secreted by a murineT cell line (clone 11.3) was measured by incubating 10⁵ T cells withvarying amounts of antigen (peptide consisting of residues 110-121 ofsperm whale myoglobin) and histocompatible spleen cells from DBA/2 mice(H-2d, 5×10⁵), which act as antigen-presenting cells (APCs), in 96 wellplates. After incubation for 24 hours at 37° C., 100 μL of thesupernatants were transferred to microtiter plates coated withcommercially available anti-gamma-IFN monoclonal antibodies (Mab)(Pharmingen, San Diego, Calif.). After incubation for an hour at roomtemperature, the plates were washed with PBS containing 1% fetal calfserum and 0.1% Tween 20, after which a second, biotinylated gamma-IFNMab was added. After a second hour of incubation, the plates were washedas before, and europium (Eu)-streptavidin (Delphia-Pharmacia) was added.Again, after an hour of incubation, an acidic buffer was added torelease Eu, which was measured by time-resolved fluorometry on a Delphiaplate reader. The amount of fluorescence was proportional to the amountof gamma-IFN that had been produced and could be quantified preciselyusing known amounts of gamma-IFN to create a standard curve.

4. Inhibition of Gamma-IFN Production by Conjugates. The ability ofPNA-polyarginine conjugates to inhibit secretion of gamma-IFN wasassayed by adding various concentrations of the above gamma-IFNconjugates with suboptimal doses of peptide antigen (0.5 AM), to amixture of clone 11.3 T cells and histocompatible spleen cells. PNAsequences lacking polyarginine moieties, and non-conjugated D-arginineheptamer, were also tested.

After 24 hours, aliquots of the cultured supernatants were taken, andthe amount of gamma-IFN was measured using the fluorescent binding assaydescribed in section 3 above. Treatment of cells with the antisensePNA-r7 conjugate resulted in an over 70% reduction in IFN secretion,whereas equivalent molar amounts of the sense PNA-r7, antisense PNAlacking r7, or r7 alone all showed no inhibition (FIG. 7).

EXAMPLE 12 Transport of Large Protein Antigen Into APCs

A conjugate of ovalbumin coupled to a poly-L-arginine heptamer wasformed by reacting a cysteine-containing polypeptide polymer(Cys-Ala-Ala-Ala-Arg₇, SEQ ID NO:20) with ovalbumin (45 kDa) in thepresence of sulfo-MBS, a heterobifunctional crosslinker (Pierce ChemicalCo., Rockford, Ill.). The molar ratio of peptide conjugated to ovalbuminwas quantified by amino acid analysis. The conjugate product wasdesignated OV-R7. The conjugate was added (final concentration ∞10 μM)to B-cells, also referred to as antigen-presenting cells (APCs), whichwere isolated according to standard methods. The APCs were incubatedwith OV-R7, and then were added to a preparation of cytotoxicT-lymphocytes isolated by standard methods. Exposure of CTLs to APCsthat had been incubated with OV-R7 produced CD8+ albumin-specific CTLs.In contrast, APCs that had been exposed to unmodified ovalbumin failedto stimulate the CTLs.

In another experiment, histocompatible dendritic cells (a specific typeof APC) were exposed to albumin-R7 conjugates and were then injectedinto mice. Subsequent analysis of blood from these mice revealed thepresence of albumin-specific CTLs. Control mice were given dendriticcells that had been exposed to unmodified albumin. The control mice didnot exhibit the albumin-specific CTL response.

EXAMPLE 13 Enhanced Uptake of V7-Derived Peptide

A conjugate consisting of a portion of the C-terminal cytoplasmic tailregion of V7 (a leukocyte surface protein) having the sequence KLSTLRSNT(SEQ ID NO:21; Ruegg et al., 1995) was synthesized with 7 arginineresidues attached to its C-terminus according to standard methods usinga peptide synthesizer (Applied Biosystems Model 433). The conjugate wasadded (final concentration ∞10 μM) to T-cells which had been isolated bystandard methods, and was incubated at 37° C. for several hours toovernight. Cells were lysed using detergent (1% Triton X-100). DNA wasremoved, and the soluble (protein-containing) fraction was subjected toimmunoprecipitation with an anti-V7 murine monoclonal antibody incombination with goat anti-mouse IgG. RAF-1 is a kinase that associateswith, and is inactivated by association with, V7.

In the absence of peptide treatment, RAF-1 protein co-precipitated withV7. In peptide-treated cells, RAF-1 protein was eliminated from the V7immunocomplex. The same peptides were unable to disrupt a complexconsisting of RAF-1 and p21 Ras, ruling out non-specific modification ofRAF-1 by the V7 peptide.

In a second study, the V7 peptide portion of the V7-poly-arginineconjugate was phosphorylated in vitro using protein kinase C. Anti-RAF-1precipitates of T cells that had been exposed to the phosphorylated V7tail peptides, but not the unphosphorylated V7 tail peptide,demonstrated potent inhibition of RAF-kinase activity.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from the spiritof the invention.

21 9 amino acids amino acid single linear peptide not provided 1 Arg LysLys Arg Arg Gln Arg Arg Arg 1 5 9 amino acids amino acid single linearpeptide not provided 2 Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 4 aminoacids amino acid single linear peptide not provided 3 Arg Arg Arg Arg 15 amino acids amino acid single linear peptide not provided 4 Arg ArgArg Arg Arg 1 5 6 amino acids amino acid single linear peptide notprovided 5 Arg Arg Arg Arg Arg Arg 1 5 7 amino acids amino acid singlelinear peptide not provided 6 Arg Arg Arg Arg Arg Arg Arg 1 5 8 aminoacids amino acid single linear peptide not provided 7 Arg Arg Arg ArgArg Arg Arg Arg 1 5 9 amino acids amino acid single linear peptide notprovided 8 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 15 amino acids aminoacid single linear peptide not provided 9 Arg Arg Arg Arg Arg Arg ArgArg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 20 amino acids amino acidsingle linear peptide not provided 10 Arg Arg Arg Arg Arg Arg Arg ArgArg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg Arg Arg 20 25 aminoacids amino acid single linear peptide not provided 11 Arg Arg Arg ArgArg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 15 Arg Arg ArgArg Arg Arg Arg Arg Arg 20 25 4 amino acids amino acid single linearpeptide not provided Other 1...4 D-Arg 12 Arg Arg Arg Arg 1 5 aminoacids amino acid single linear peptide not provided Other 1...5 D-Arg 13Arg Arg Arg Arg Arg 1 5 6 amino acids amino acid single linear peptidenot provided Other 1...6 D-Arg 14 Arg Arg Arg Arg Arg Arg 1 5 7 aminoacids amino acid single linear peptide not provided Other 1...7 D-Arg 15Arg Arg Arg Arg Arg Arg Arg 1 5 8 amino acids amino acid single linearpeptide not provided Other 1...8 D-Arg 16 Arg Arg Arg Arg Arg Arg ArgArg 1 5 9 amino acids amino acid single linear peptide not providedOther 1...9 D-Arg 17 Arg Arg Arg Arg Arg Arg Arg Arg Arg 1 5 10 basepairs nucleic acid single linear Other not provided Other 1...10 wherethe bonds are peptide nucleic acid bonds 18 AACGCTACAC 10 10 base pairsnucleic acid single linear Other not provided Other 1...10 where thebonds are peptide nucleic acid bonds 19 GTGTAGCGTT 10 11 amino acidsamino acid single linear peptide not provided 20 Cys Ala Ala Ala Arg ArgArg Arg Arg Arg Arg 1 5 10 16 amino acids amino acid single linearpeptide not provided 21 Lys Leu Ser Thr Leu Arg Ser Asn Thr Arg Arg ArgArg Arg Arg Arg 1 5 10 15

What is claimed is:
 1. A composition for delivering paclitaxel across abiological membrane, comprising: a conjugate containing paclitaxelcovalently attached via a linker that is cleavable in vivo to atransport peptide, wherein said peptide consists of from 6 to 25 aminoacid residues, at least 50% of which contain a guanidino or amidinosidechain moiety, and contains at least 6 contiguous guanidino and/oramidino sidechain moieties, whereby said attached transport peptide iscapable of delivering said paclitaxel across a biological membrane. 2.The composition of claim 1, wherein at least 70% of the residues in thepeptide contain a guanidino sidechain moiety.
 3. The composition ofclaim 1, wherein no guanidino or amidino sidechain moiety is separatedfrom another such moiety by more than one non-guanidino or non-amidinoresidue.
 4. The composition of claim 1, wherein said peptide consists offrom 7 to 20 residues.
 5. The composition of claim 1, wherein eachresidue contains a guanidino group.
 6. The composition of claim 1,wherein said transport peptide contains at least six contiguous arginineresidues.
 7. The composition of claim 6, wherein sad peptide consists offrom 7 to 20 residues.
 8. The composition of claim 6, wherein all ofsaid residues are arginine residues.
 9. The composition of claim 6,wherein at least one of said arginine residues has a D-configuration.10. The composition of claim 6, wherein all of said arginine residueshave a D-configuration.
 11. The composition of claim 1, wherein saidpeptide is covalently attached via C7-oxygen of said paclitaxel.
 12. Thecomposition of claim 1, wherein said peptide is covalently attached viaC2′-oxygen of said paclitaxel.
 13. The composition of claim 1, whereinsaid cleavable linker contains an ester group.
 14. The composition ofclaim 1, wherein said cleavable linker contains a disulfide group. 15.The composition of claim 1, wherein said cleavable linker is aphotolabile linker.